Abstract

We investigated the role of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor in the induction and expression of an amphetamine-induced conditioned place preference (CPP) in mice. The selective AMPA-receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX) failed to prevent the induction of a CPP, except at a dose (30 mg/kg) that also produced a conditioned place aversion. NBQX also failed to affect the expression of a CPP at a dose high enough to reduce activity levels. In contrast, the less selective AMPA receptor antagonist 6-cyano-7-nitroquinoxalone-2,3-dione (CNQX) prevented the expression of a CPP at doses (1–10 mg/kg) that had no effect on activity levels. We therefore tested the possibility that CNQX exerted its effects due to antagonism at the glycine site of theN-methyl-d-aspartate receptor. The glycine-site antagonist 7-chloro-4-hydroxy-3-(2-phenoxy)phenyl-2(1H)-quinolone also prevented the expression of a CPP at doses that had no effect on activity levels (0.1–0.3 mg/kg). These results suggest that neither the induction nor the expression of an amphetamine-induced CPP requires AMPA receptor-mediated transmission and that effects found in previous studies using the less selective AMPA receptor antagonists may be due to the effects of these compounds at the glycine site of the N-methyl-d-aspartate receptor.

The conditioned place preference (CPP) paradigm has frequently been used as a method of assessing the reinforcing properties of drugs of abuse and to study the neural mechanisms underlying conditioned reinforcement. The majority of drugs abused by humans, including psychostimulants, opiates, ethanol, and benzodiazepines, will also produce a CPP (see,Carr et al., 1989, for review). During recent years, it has been reported that both the development and expression of a CPP are dependent on glutamatergic transmission. Specifically, it has been proposed that there are different roles for two of the main receptor subtypes for glutamate: theN-methyl-d-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors (Cervo and Samanin, 1995; Tzschentke and Schmidt, 1997).

Transmission via the NMDA receptor subtype has been argued to be necessary for both the induction and the expression of a CPP because the NMDA channel blocker MK-801 prevented the induction of a cocaine (Cervo and Samanin, 1995), methamphetamine (Kim and Jang, 1997), and morphine CPP in rats (Tzschentke and Schmidt, 1995, 1997; Kim et al., 1996). This effect has been replicated with the competitive NMDA receptor antagonist CGP37849, preventing the induction of a morphine CPP, again in rats (Tzschentke and Schmidt, 1995). However, Hoffman (1994) reported that MK-801 failed to affect the induction of an amphetamine (AMPH)-induced CPP. Studies investigating the effect of NMDA receptor antagonists on the expression of a CPP reveal conflicting results. MK-801 failed to prevent the expression of a cocaine-induced CPP (Cervo and Samanin, 1995) but did prevent the expression of a morphine-induced CPP (Tzschentke and Schmidt, 1997). Furthermore,Bespalov (1996) reported that the competitive antagonist (±)-3-(2-carboxy-piperazine-4-yl)-propyl-1-phosphonic acid prevented the expression of an AMPH-induced CPP.

The underlying involvement of AMPA receptors in CPP appears to be much clearer. First, the AMPA receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) prevented the expression of a CPP induced by cocaine (Cervo and Samanin, 1995; Kaddis et al., 1995), AMPH, and morphine (Layer et al., 1993). Second, the noncompetitive AMPA receptor antagonist GYKI 52466 has been reported to prevent the expression of both a morphine and an AMPH-induced CPP (Tzschentke and Schmidt, 1997). Although DNQX infused into the nucleus accumbens was shown to be ineffective against the induction of a morphine CPP (Layer et al., 1993) and intracerebroventricular DNQX was ineffective against the induction of a cocaine-induced CPP (Cervo and Samanin, 1995), intra-accumbens DNQX did attenuate the induction of an AMPH (Layer et al., 1993) and a cocaine-induced CPP (Kaddis et al., 1995). Therefore, the current literature suggests that AMPA receptors mediate the expression of drug-induced CPPs but that AMPA receptor antagonists show a more inconsistent profile when tested on the induction.

The purpose of the studies reported here was to assess the role of glutamatergic transmission via AMPA receptors in an AMPH-induced CPP in mice using the selective competitive AMPA receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f) quinoxaline (NBQX) (Sheardown et al., 1990). For comparison, we also investigated the effect of the less selective AMPA receptor antagonist 6-cyano-7-nitroquinoxalone-2,3-dione (CNQX), which has a similar pharmacological profile to DNQX (Birch et al., 1988; Harris and Miller, 1989; Kessler et al., 1989; Pellegrini-Giampietro et al., 1989), although its selectivity for AMPA receptors compared with NMDA receptors is slightly higher than that for DNQX (Sheardown et al., 1990). Subsequently, we explored the involvement of the glycine site of the NMDA receptor by assessing the effect of the strychnine-insensitive glycine-site antagonist L-701,324. The results of these studies allowed us to make conclusions concerning the reliability of the previous studies investigating AMPA receptor involvement in a drug-induced CPP.

Materials and Methods

Animals

Male C57Bl/6J × SV129 mice, bred at the University of Sussex, were used in all experiments. Animals weighed 21 to 29 g at the start of experiments and were housed in groups of two or three under a 12:12-h light/dark cycle (lights on at 7:00 AM) with ad libitum access to food and water. Holding room temperature was maintained at 20–21°C, and humidity was 40 to 60%. Experiments were performed between 8:30 AM and 3:00 PM. All experiments were carried out under U.K. legislation on animal experimentation.

Apparatus

All experiments were performed in a three-compartment place conditioning apparatus. The two outer compartments (200 × 200 × 200 mm) were separated by a central compartment (200 × 50 × 200 mm) and differed in visual and tactile cues. One outer compartment had white walls with a perforated metal floor, whereas the other had black and white walls (each wall was split along the diagonal, with the top half painted black and the bottom half painted white) with a smooth clear Perspex floor. The central compartment allowed movement from one compartment to the other and could also be blocked from the outer compartments with clear doors, to restrict animals to a particular compartment during the conditioning phase. The movement and location of animals during the preconditioning and test phases were recorded using a video camera (Sony SPT-M108CE) connected to a video cassette recorder (Panasonic AG-5700) to allow subsequent analysis of preference and activity.

Procedure

Four separate experiments were performed, for which the basic protocol was identical. In all experiments, we used an AMPH-induced (0.25 mg/kg) CPP as the paradigm to assess the role of glutamatergic mechanisms in conditioned reinforcement. In experiments 1 and 2, we investigated the effect of NBQX on the induction and the expression of a CPP. In experiments 3 and 4, we investigated the effect of CNQX and 7-chloro-4-hydroxy-3-(2-phenoxy)phenyl-2(1H)-quinolone (L-701,324) on the expression of a CPP. The design of these experiments is shown in Table 1.

CPP Procedure

The CPP procedure took place over 10 days and consisted of the following three phases.

Preconditioning Phase (Day 1).

Animals received vehicle (t = −20 min), followed by saline (t = 0). Consequently, they were placed in the CPP apparatus and allowed to explore freely for 10 min. The behavior of the animals was recorded for subsequent analysis. These data provided the baseline preferences for the two compartments. Initial preferences for the two compartments did not differ significantly (mean ± S.E.M. time, 199 ± 3.7 s in compartment A, 201 ± 4.1 s in compartment B during a 10-min test, calculated from all experiments).

Conditioning Phase (Days 2–9).

On every second day, in experiment 1, animals received NBQX (vehicle, 3, 10, or 30 mg/kg) 20 min before AMPH or NBQX (vehicle, 30 mg/kg) 20 min before saline, immediately before confinement to one compartment of the CPP apparatus. On every second day, in experiments 2, 3, and 4, animals received vehicle 20 min before AMPH and confinement to one compartment. In all four experiments, on alternate days, all animals received vehicle 20 min before saline and confinement to the other compartment. Confinement to compartments lasted for 20 min. Assignment of drug-paired compartments was performed randomly and was fully counterbalanced across groups.

Drugs

d-AMPH sulfate was obtained from Sigma Chemical (Poole, Dorset, UK). NBQX and CNQX were donated by Schering AG (Berlin, Germany). L-701,324 was donated by Merck Sharp and Dohme (Essex, UK). AMPH was dissolved in 0.9% saline. NBQX was dissolved in 1 M sodium hydroxide, and 0.1 M hydrochloric acid was added to take the pH back toward neutral. The solution was made up to its final volume with distilled water. This resulted in drug solutions of pH 9 to 10. NBQX vehicle was pH matched to drug by adding 0.1 M sodium hydroxide to distilled water. CNQX and L-701,324 were suspended in 0.3% Tween-80 in distilled water, which was also used as the vehicle. All drugs were administered i.p. at an injection volume of 10 ml/kg. All doses refer to drug base.

Data Analysis

The time spent in each compartment and activity within the compartments were scored blind. An animal was considered to be in a compartment only when its entire body was within that compartment. Activity was measured as line crossings of two perpendicular lines crossing the midpoint and extending to the walls of the compartment. For analysis of place conditioning, the time spent in the drug-paired compartment minus the time spent in the vehicle-paired compartment was used as the dependent variable. For analysis of activity levels, total line crossings in both compartments were used as the dependent variable. Data for place conditioning were initially analyzed using a two-way mixed-factor ANOVA (except for test 1 in experiment 4), with test session (preconditioning and test phase) and treatment as independent variables. After a significant interaction, a one-way between-subject ANOVA was performed on the test session data only. If this produced a significant effect of treatment, post hoc analysis was performed using the Duncan’s test. Due to the sedative effect of the higher dose range of L-701,324 (experiment 4, test 1), the variance between groups was heterogeneous, and the data were not suitable for parametric analysis. For this reason, a comparison of the preference during the test phase and the preconditioning phase for each group was made using Wilcoxon’s signed rank test to deduce whether conditioning had occurred. Data for activity levels were analyzed using a one-way ANOVA on activity levels during the test phase, followed by Duncan’s test if a significant effect of treatment was found. All statistical analysis was performed using the SPSS statistical package for Windows (Version 6.1, SPSS Inc., Chicago, IL).

Results

CPP.

The results from experiment 1, showing the effect of NBQX on the induction of place conditioning, are shown in Fig.1. Analysis revealed a significant interaction between treatment and test session (F5,53 = 4.90, p < .01). The one-way ANOVA on the preference for the AMPH-paired compartment during the test phase revealed a significant effect of treatment (F5,53 = 12.1,p < .001), indicating that significant place conditioning had taken place. Post hoc comparisons showed a significant effect of AMPH compared with saline, indicating place conditioning (p < .05). NBQX, at doses of 3 and 10 mg/kg, failed to affect the induction of place conditioning, whereas a dose of 30 mg/kg significantly attenuated the preference (p < .05). NBQX, at a dose of 30 mg/kg, also produced a significant place aversion, compared with saline controls (p < .05).

The results from experiment 2, showing the effect of NBQX on the expression of AMPH-induced place conditioning, are shown in Fig.2. The two-way ANOVA on treatment by test session revealed a significant interaction (F3,31 = 3.88, p = .018) and a subsequent one-way ANOVA on test-phase data revealed a significant effect of treatment (F3,31= 3.53, p = .026). Post hoc analysis showed that groups conditioned to AMPH displayed a significant place preference compared with saline-conditioned groups (p < .05). NBQX at 10 mg/kg had no effect on this place conditioning.

The effect of CNQX on the expression of an AMPH-induced CPP (experiment 3) is shown in Fig. 3. A two-way ANOVA on treatment by test session revealed a significant interaction (F5,52 = 3.35, p = .011), and a subsequent one-way ANOVA on test-phase data revealed a significant effect of treatment (F5,52= 3.25, p = .013). Post hoc analysis again showed significant place conditioning, with AMPH treatment resulting in more time spent in the drug compartment than saline treatment (p < .05). CNQX, at doses ranging from 1 to 10 mg/kg, blocked the expression of this CPP (p < .05).

Figure 4 shows the results of experiment 4, in which the effect of the higher dose range of L-701,324 on the expression of an AMPH-induced CPP was assessed. The extreme variation observed in groups treated with L-701,324 was due to a large spread in the preference scores, which appeared to occur as a result of the locomotor suppressant effects of the compound. Wilcoxon’s signed rank test revealed that only the group conditioned to AMPH and tested with L-701,324 vehicle displayed significant place conditioning (z = −2.80, p < .01). Groups conditioned to AMPH and tested with L-701,324 (1–10 mg/kg) failed to exhibit place conditioning.

The effects of the lower dose range of L-701,324 on the expression of an AMPH-induced CPP are shown in Fig. 5. Two-way ANOVA revealed a significant treatment by test-session interaction (F5,54 = 5.19,p < .01), and subsequent ANOVA on the test-session data revealed a significant effect of treatment (F5,54 = 4.49, p < .01). Post hoc analysis revealed that AMPH produced a significant CPP (p < .05), which was prevented by L-701, 324 at doses of 0.1 and 0.3 mg/kg. A dose of 0.03 mg/kg L-701,324 failed to prevent the expression of the AMPH CPP.

Discussion

The studies reported here show that the selective, competitive AMPA receptor antagonist NBQX does not prevent the induction of an AMPH-induced CPP, except at a dose that also produces a significant conditioned place aversion (CPA). NBQX also had no effect on the expression of an AMPH-induced CPP at a dose high enough to cause a significant reduction in activity. Conversely, both the less selective AMPA receptor antagonist CNQX and the glycine-site antagonist L-701,324 prevented the expression of an AMPH-induced CPP at doses that did not affect activity levels. A summary of the relative affinities of the tested compounds for the AMPA receptor and glycine site of the NMDA receptor is shown in Table 2.

Affinities of NBQX, CNQX, and L-701,324 for AMPA receptors and the glycine site of the NMDA receptor

The fact that NBQX affected the induction of a CPP only at a dose that also produced a CPA suggests that AMPA receptors do not mediate the induction of an AMPH-induced CPP (experiment 1). The attenuation of the CPP seen at a dose of 30 mg/kg NBQX is most likely explained by the aversive properties of the drug, as shown by the production of a CPA, counteracting the reinforcing effect of AMPH. This result is consistent with previous studies that reported that DNQX did not prevent the induction of a CPP to cocaine and morphine (Layer et al., 1993; Cervo and Samanin, 1995). However, there also are reports of intra-accumbens DNQX preventing the induction of a CPP to cocaine (Kaddis et al., 1995) and AMPH (Layer et al., 1993). A potential problem with these studies is that neither included a control group that received DNQX alone during the conditioning phase. As experiment 1 shows, an AMPA receptor antagonist is capable of producing a marked CPA, and therefore one possible explanation for the apparent effect of DNQX is that the antagonist possesses aversive properties that counteract the reinforcing properties of cocaine or AMPH. In line with this possibility, both studies reported an attenuation of the CPP due to DNQX, rather than a complete blockade, which is very similar to the effect seen in the experiment reported here, at the 30 mg/kg dose of NBQX.

The fact the NBQX did not prevent the expression of the CPP at a dose (10 mg/kg) that caused a significant, although small, reduction in activity levels suggests that AMPA receptors do not mediate the expression of an AMPH-induced CPP (experiment 2). Although this study involved the use of a single dose of NBQX, testing of higher doses was not possible because of profound disruption of locomotor activity. We have shown previously that NBQX is behaviorally active in the mouse strain used in this experiment at doses as low as 3 mg/kg i.p. (Mead and Stephens, 1998). There also is evidence to suggest that NBQX provides an effective block of AMPA receptors in mice at doses lower than 10 mg/kg (Steppuhn and Turski, 1993; Swedberg et al., 1995). Finally, analysis of activity levels during the test phase of this experiment revealed that NBQX was behaviorally active, in that it produced a significant reduction in activity levels.

The results from experiment 2 stand in apparent contrast to a number of reports of AMPA receptor antagonists preventing the expression of a CPP to morphine, AMPH, and cocaine (Layer et al., 1993; Cervo and Samanin, 1995; Kaddis et al., 1995). To our knowledge, there are no previous reports of AMPA receptor antagonists failing to prevent the expression of a drug-induced CPP. One possible explanation of the contradiction between the current study and previous work is that although this study used mice, all previous studies have used rats. A second possibility is that the differences are due to the selectivity of the antagonist used. All reports to date revealing an attenuation or a prevention of the expression of a CPP have involved the use of DNQX or GYKI 52466. Both of these compounds have an influence on NMDA-mediated transmission. DNQX has significant affinity for the glycine site of the NMDA receptor (Kessler et al., 1989; Pellegrini-Giampietro et al., 1989; Sheardown et al., 1990; Goldstein and Litwin, 1993; Yoneda et al., 1993; Birch et al., 1988), whereas GYKI 52466 shows functional antagonism of NMDA-induced seizures (Steppuhn and Turski, 1993). Both of these possibilities were examined in experiments 3 and 4.

Experiment 3 revealed that the less selective AMPA receptor antagonist CNQX prevented the expression of an AMPH-induced CPP at doses devoid of effects on activity. CNQX is an antagonist with a similar pharmacological profile to DNQX (Birch et al., 1988; Harris and Miller, 1989; Kessler et al., 1989; Pellegrini-Giampietro et al., 1989), although its selectivity for AMPA receptors is slightly higher (Sheardown et al., 1990). First, the fact that an effect of CNQX was seen in our experiment at doses (1–10 mg/kg i.p) that did not affect activity levels argues against the possibility of a species difference, as the effect of CNQX in mice was consistent with effects of DNQX in rats. Second, it suggests that the route of drug administration was not a decisive factor in explaining differences in published results. The previous reports on AMPA receptor antagonists preventing the expression of CPP various routes of administration used, including intra-accumbens, intracerebroventricular, and systemic. The present study confirms that the less selective AMPA receptor antagonists prevent the expression of drug-induced CPPs when given systemically. This appears to suggest that the reason for the different results found with NBQX and the other AMPA receptor antagonists (DNQX, CNQX, and GYKI 52466) is that the latter substances influence NMDA receptor-mediated transmission. In particular, the affinity of CNQX and DNQX for the glycine site of the NMDA receptor may be important for their activity in CPP experiments. We therefore predict that a glycine-site antagonist would also prevent the expression of an AMPH-induced CPP.

The results of experiment 4 provide evidence in support of this explanation. The selective glycine site antagonist L-701,324 prevented the expression of an AMPH-induced CPP, at doses that did not affect activity levels. The results of test 1 were complicated by the sedative effects of the higher dose range of L-701,324 (1–10 mg/kg), although analysis revealed that these doses prevented the expression of the CPP. Test 2, however, revealed a dose-dependent effect of L-701,324 on the expression of the CPP, with the two higher doses (0.1 and 0.3 mg/kg i.p.) preventing the CPP, whereas a dose of 0.03 mg/kg reduced the CPP but not significantly. All three of these lower doses did not affect activity levels. Recently, a similar effect of L-701,324 has been reported by Danysz et al. (1998), whereby an attenuation in the expression of a morphine-induced CPP in rats was observed.

The results of the present study suggest that previous findings with the less selective AMPA receptor antagonists may be misleading. We report here that the effects of an antagonist such as CNQX differ from those found with the more selective antagonist NBQX. Furthermore, we report that this difference can be explained by the action of CNQX at the modulatory strychnine-insensitive glycine site of the NMDA receptor, as an antagonist at this site shows a similar behavioral profile to CNQX. The implications of this study are not necessarily restricted to the AMPH-CPP paradigm. Further work is necessary to see whether this finding extends to other drugs of abuse that produce a CPP, such as morphine and cocaine, although the report by Danysz et al. (1998) suggests that this may be the case. Perhaps more importantly, care must be taken in interpreting previous in vivo studies using the class of less selective AMPA receptor antagonists in other paradigms.

It has been suggested that AMPA receptors play an important role in mediating AMPH-enhanced responding for conditioned reinforcement (Burns et al., 1994) and responding for a drug-paired stimulus (Hitchcott and Phillips, 1997). Both of these studies involved the use of CNQX as a method of investigating AMPA receptor involvement in the behavioral response. Similarly, many reports of drug-induced behavioral sensitization have also used CNQX and DNQX as tools for studying AMPA receptor involvement in the behavioral effects of drugs of abuse (Karler et al., 1991, 1994; Pierce et al., 1996). Our present findings suggest it may be necessary to reexamine the conclusions of these studies if more selective antagonists give rise to different effects.

In summary, the selective competitive AMPA receptor antagonist NBQX did not affect either the induction or the expression of an AMPH-induced CPP. In contrast, both the less selective AMPA receptor antagonist CNQX and the selective glycine-site antagonist L-701,324 prevented the expression of an AMPH-induced CPP. These results suggest that the induction and the expression of an AMPH-induced CPP in mice do not depend on glutamatergic transmission via AMPA receptors but that the expression of the response is dependent on modulation of NMDA receptor-mediated transmission via the glycine site. This importance of the glycine site of the NMDA receptor in mediating drug-induced conditioned reinforcement suggests that compounds acting at this site may have therapeutic potential in the treatment of drug craving. The present study also raises doubts concerning the conclusions drawn from previous studies using the less selective AMPA receptor antagonists, such as DNQX and CNQX. Further work is necessary to deduce whether the conclusions drawn from these studies are indeed misleading.

Acknowledgments

We thank Dr. E. Ottow (Schering AG) for a generous gift of NBQX and CNQX and Dr. P. Hutson (Merck Sharp and Dohme) for the generous donation of L-701,324.

(1998) AMPA receptors are involved in the expression of amphetamine-induced behavioural sensitisation, but not in the expression of amphetamine-induced conditioned activity in mice.Neuropharmacology37:1131–1138.